The long-range goal of this laboratory is to understand the functional interactions between a tumor virus and the host cell. The approach has been to define the biological functions expressed by the SV40 transforming protein, large tumor antigen (T-ag), in infected and transformed cells. Our efforts during the previous funding period focused on characterizing the plasma membrane-associated form of SV40 T-ag and analyzing a viral mutant that encoded a cytoplasmic T-ag (cT-ag) defective for nuclear transport. We showed that surface T-ag is associated with host protein p53 in the membrane of transformed cells, that the complex is rapidly turned over in the membrane, that the conformation in the membrane exposes both amino and carboxy termini of T-ag on the exterior of the cell, and that the expression of surface T-ag correlates with cell growth. Studies of the nuclear-transport-defective viral mutant succeeded in identifying the single amino acid change responsible for blocking nuclear transport of T-ag. Finally, we found that T-ag is modified by glycosylation. These efforts culminated in a perspective that forms the basis of the current proposal, namely that the surface-associated form of SV40 T-ag is functionally important. Proposed objectives are logical extensions of our previous studies and are timely in the context of current thinking regarding possible molecular mechanisms in carcinogenesis. The following specific aims are proposed. (1) To categorize into complementation groups the transformation-related functions expressed by T-ag. The approach will include co-transfection/transformation assays using the mutant cT-ag-encoding plasmid and known cloned oncogenes in primary and immortalized host cells. (2) To further examine the observed relationship between surface T-ag expression and cell growth. (3) To test the hypothesis that surface-associated T-ag may be functioning in a growth factor pathway by mimicking an "activated" growth factor receptor. Internalization of surface T-ag and processing by lysosomal proteases will be sought, and the possible requirement of complex formation with cellular protein p53 to generate an "active" T-ag conformation examined. (4) To further characterize the glycosylation modification of T-ag, especially with respect to mapping the glycosylation sites on the polypeptide. T-ag may be a good model for O-glycoproteins in general. Finally, (5) To study the intracellular trafficking of T-ag. Special emphasis will be placed on determining the sites of T-ag synthesis and O-glycosylation in the cell, as well as investigating the sorting that separates T-ag molecules destined for nuclear and surface localizations. These studies should yeild important insights into the biological functioning of a prototype viral transforming protein and broaden our understanding of molecular mechanisms involved in cellular transformation.